Compositions for Fecal Floral Transplantation and Methods for Making and Using Them and Devices for Delivering Them

ABSTRACT

In alternative embodiments, the invention provides compositions, e.g., formulations, used for gastric, gastrointestinal and/or colonic treatments or lavage, e.g., orthostatic lavage, e.g., for inducing the purgation (e.g., cleansing) of a gastrointestinal (GI) tract, including a colon; and methods for making and using them. In alternative embodiments, compositions and methods of the invention are used for the stabilization, amelioration, treatment and/or prevention of constipation, for the treatment of abdominal pain, particularly non-specific abdominal pain, and diarrhea, including diarrhea caused by a drug side effect, a psychological condition, a disease or a condition such as Crohn&#39;s Disease, a poison, a toxin or an infection, e.g., a toxin-mediated traveler&#39;s diarrhea, or  C. difficile  or the pseudo-membranous colitis associated with this infection. In alternative embodiments, the invention provides pharmaceuticals and products (articles) of manufacture for delivering these compositions and formulations to an individual, e.g., a human or an animal. The invention also provides devices for delivering a fecal material to a patient.

TECHNICAL FIELD

This invention generally relates to medicine and gastroenterology,pharmacology and microbiology. In alternative embodiments, the inventionprovides compositions, e.g., formulations or preparations, and devices,used for the transplantation of a treated or isolated fecal flora, andmethods for making and using them. In alternative embodiments,compositions, devices and methods of the invention can be used for anygastric, gastrointestinal and or colonic treatment or lavage. Inalternative embodiments, compositions, devices and methods of theinvention are used for the amelioration, stabilization, treatment and/orprevention of a disease or a condition such as constipation, Crohn'sDisease, exposure to a poison or a toxin or for an infection, e.g., atoxin-mediated travelers diarrhea; or any bowel disease or conditionhaving a bowel dysfunction component, for example, an inflammatory boweldisease (IBD), Crohn's disease, hepatic encephalopathy, enteritis,colitis, irritable bowel syndrome (IBS), fibromyalgia (FM), chronicfatigue syndrome (CFS), depression, attention deficit/hyperactivitydisorder (ADHD), multiple sclerosis (MS), systemic lupus erythematosus(SLE), travelers' diarrhea, small intestinal bacterial overgrowth,chronic pancreatitis, or a pancreatic insufficiency. In alternativeembodiments, the invention provides pharmaceuticals and products(articles) of manufacture for delivering these compositions andformulations to an individual, e.g., a human or an animal. The inventionalso provides devices for delivering a fecal material to an individual,e.g., a patient.

BACKGROUND

Bacterial flora of the bowel has recently gained importance from atherapeutic point of view. It is now realized that the human flora,rather than just being waste material resulting from digestion of food,is an important virtual organ containing large numbers of livingmicroorganisms. There are in excess of one hundred thousand differentsubspecies—or more—arranged in families and subgroups of geneticallydifferent but often linearly related organisms. The waste “material”makes up a proportion of the flora. The bacterial content of the florais actively breaking down or metabolizing the non-absorbed matter,largely fiber, on which the bacterial cells grow. Because the bacterialflora is contained within the human body and is made up of livingcomponents it constitutes in fact as a living organ or a virtual organ.

This virtual organ can be healthy in that it doesn't contain anypathogenic organisms, or it can become infected or infested withparasite, bacteria or viruses. When infected with some pathogenicspecies, such infecting species can manufacture molecules that affectsecretion, which can cause pain, or can paralyze the bowel causingconstipation. Infection of the bowel flora or bowel flora organ canimpact the health of the individual.

Many of these infections can be acute, such as cholera, but some can bechronic and can really impact on the life of the individual carrying theinfected flora. For example, after antibiotic therapy some of thefamilies of the bacteria can be suppressed or eradicated and infectiousagents such as Clostridium difficile and other pathogens can lodge andbecome passengers within the human flora. These ‘passengers’ are alsopathogenic because they can produce toxins e.g. toxins A and B for C.difficile.

Definitions

The following are some definitions that may be helpful in understandingthe description of the present invention. These are intended as generaldefinitions and should in no way limit the scope of the presentinvention to those terms alone, but are put forth for a betterunderstanding of the following description.

Unless the context requires otherwise or specifically stated to thecontrary, integers, steps or elements of the invention recited herein assingular integers, steps or elements clearly encompass both singular andplural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated step or element orinteger or group of steps or elements or integers, but not the exclusionof any other step or element or integer or group of elements orintegers. Thus, in the context of this specification, the term“comprising” means “including principally, but not necessarily solely”.

SUMMARY

According to a first aspect of the present invention, there is provideda delivery vehicle, formulation, composition, pharmaceuticalpreparation, product of manufacture, container or device, comprising: anentire (or substantially entire) microbiota; a treated or untreatedfecal flora; a complete or partial fecal flora, a fecal florasubstantially or completely purified of non-fecal floral fecal material,or a partially, substantially or completely isolated or purified fecalflora, made by a process comprising:

(i) providing an entire (or substantially entire) microbiota, a treatedor untreated fecal flora sample, a complete or partial fecal florasample, a fecal flora substantially or completely purified of non-fecalfloral fecal material or a partially, substantially or completelyisolated or purified fecal flora; and, a delivery vehicle, formulation,pharmaceutical preparation, composition, product of manufacture,container or device, and

(ii) placing the entire (or substantially entire) microbiota, thetreated or untreated fecal flora sample, the complete or partial fecalflora sample, the fecal flora substantially or completely purified ofnon-fecal floral fecal material, or the partially, substantially orcompletely isolated or purified fecal flora in the delivery vehicle,formulation, composition, pharmaceutical preparation, product ofmanufacture, container or device.

According to a second aspect of the present invention, there is provideda product (article) of manufacture comprising a delivery vehicle,formulation, composition pharmaceutical preparation, container or deviceof the first aspect.

According to a third aspect of the present invention, there is provideda method for making a delivery vehicle, formulation, compositionpharmaceutical preparation, product of manufacture, container or deviceaccording to the first or second aspect comprising

(i) providing: an entire (or substantially entire) microbiota; a treatedor untreated fecal sample; a complete or partial fecal flora sample, afecal flora substantially or completely purified of non-fecal floralfocal material or a partially, substantially or completely isolated orpurified fecal flora; and, a delivery vehicle, formulation,pharmaceutical preparation, composition product of manufacture,container or device, and

(ii) placing the entire (or substantially entire) microbiota, treated oruntreated fecal sample, the complete or partial fecal flora, the fecalflora substantially or completely purified of non-fecal floral fecalmaterial or the partially, substantially or completely isolated orpurified fecal flora in the delivery vehicle, formulation,pharmaceutical preparation, composition, product of manufacture,container or device, and creating a substantially or completelyoxygen-free environment in the container or device.

According to a fourth aspect of the present invention there is provideda method for the amelioration, stabilization, treatment and/orprevention of an infection, disease, treatment, poisoning or a conditionhaving a bowel dysfunction component or side-effect, or for theamelioration, treatment and/or prevention of a constipation, for thetreatment of an abdominal pain, a non-specific abdominal pain or adiarrhea, a diarrhea caused by: a drug side effect or a psychologicalcondition or Crohn's Disease, a poison, a toxin or an infection, atoxin-mediated traveler's diarrhea, or a Clostridium or a C. perfringenswelchii or a C. difficile infection or a pseudo-membranous colitisassociated with a Clostridium infection, comprising:

administering to an individual in need thereof via a delivery vehicle,formulation, composition, pharmaceutical preparation, product ofmanufacture, container or device of the first aspect, or a product(article) of manufacture of the second aspect the entire (orsubstantially entire) microbiota, the treated or untreated fecal florasample, the complete or partial fecal flora sample, the fecal florasubstantially or completely purified of non-fecal floral fecal material,or the partially, substantially or completely isolated or purified fecalflora.

According to a fifth aspect of the present invention, there is provideda delivery vehicle, formulation, composition, pharmaceuticalpreparation, product of manufacture, container or device comprising:

an entire (or substantially entire) microbiota; a partially,substantially or completely isolated or purified fecal flora; or, acomposition comprising a fecal flora substantially or a completelypurified of non-fecal floral fecal material.

According to a sixth aspect of the present invention, there is provideda method for the amelioration, stabilization, treatment and/orprevention of an infection, disease, treatment, poisoning or a conditionhaving a bowel dysfunction component or side-effect comprisingadministering to an individual in need thereof via a delivery vehicle,formulation, composition, pharmaceutical preparation, product ofmanufacture, container or device according to the fifth aspect theentire (or substantially entire) microbiota, the partially,substantially or completely isolated or purified fecal flora, or thecomposition comprising a fecal flora substantially or a completelypurified of non-fecal floral fecal material.

According to a seventh aspect of the present invention, there isprovided a device for delivering a fecal material or a composition ofthe first aspect, comprising:

(a) a device as illustrated in FIG. 1B or FIG. 2; or

(b) a device comprising

(i) a bag or container comprising an exit aperture operably connected tothe proximal end of a flexible tube or equivalent,

(ii) an open or close valve or equivalent or an obdurator screwtop atthe distal end of the flexible tube or equivalent, and

(iii) a pump, or a hand pump, for moving material in the bag orcontainer through the flexible tube or equivalent and out the distal endor out the open or close valve or equivalent; or (c) the device of (a)or (b), further comprising a fecal material or a composition of thefirst aspect.

According to an eighth aspect of the present invention, there isprovided a bag or container comprising: an entire (or substantiallyentire) microbiota; a treated or untreated fecal flora; a complete orpartial fecal flora, a fecal flora substantially or completely purifiedof non-fecal floral material or, a partially, substantially orcompletely isolated or purified fecal flora, or a composition thereofwherein the bag or container is structurally the same as or similar to abag or container of a device of the seventh aspect.

According to a ninth aspect of the present invention, there is provideda method for the amelioration, stabilization, treatment and/orprevention of, or decreasing or delaying the symptoms of, an infection,disease, treatment, poisoning or a condition having a bowel dysfunctioncomponent or side-effect, or for the amelioration, treatment and/orprevention of a constipation, for the treatment of an abdominal pain, anon-specific abdominal pain or a diarrhea, a diarrhea caused by: a drugside effect or a psychological condition or Crohn's Disease, a poison, atoxin or an infection, a toxin-mediated traveler's diarrhea, or aClostridium or a C. perfringens welchii or a C. difficile so infectionor a pseudo-membranous colitis associated with a Clostridium infection,or for preventing, or decreasing or delaying the symptoms of, orameliorating or treating individuals with spondyloarthropatliy,spondylarthritis or sacrolileitis (an inflammation of one or bothsacroiliac joints); a nephritis syndrome; an inflammatory or anautoimmune condition having a gut or an intestinal component; lupus;irritable bowel syndrome (IBS or spastic colon); or a colitis;Ulcerative Colitis or Crohn's Colitis; constipation; autism; adegenerative neurological diseases; amyotrophic lateral sclerosis (ALS),Multiple Sclerosis (MS) or Parkinson's Disease (PD); a MyoclonusDystonia; Steinert's disease; proximal myotonic myopathy; an autoimmunedisease; Rheumatoid Arthritis (RA) or juvenile idiopathic arthritis(JIA); Chronic Fatigue Syndrome; benign myalgic encephalomyelitis;chronic fatigue immune dysfunction syndrome; chronic infectiousmononucleosis; epidemic myalgic encephalomyelitis; obesity;hypoglycemia, pre-diabetic syndrome, type I diabetes or type IIdiabetes; Idiopathic thrombocytopenic purpura (ITP); an acute or chronicallergic reaction; hives, a rash, a urticaria or a chronic urticaria;and/or insomnia or chronic insomnia, Grand mal seizures or petit malseizures, comprising:

administering to an individual in need thereof via a delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device of the first aspect or a product (article) ofmanufacture of the second aspect of the entire (or substantially entire)microbiota, the treated or untreated fecal flora sample, the complete orpartial fecal flora sample, the fecal flora substantially or completelypurified of non-fecal floral fecal material, or the partially,substantially or completely isolated or purified fecal flora, in single,repeat or multiple administrations, deliveries or infusions.

According to a tenth aspect of the present invention, there is providedan entire (or substantially entire) microbiota, a treated or untreatedfecal flora sample, a complete or partial fecal flora sample, a fecalflora substantially or completely purified of non-fecal floral fecalmaterial, or a partially, substantially or completely isolated orpurified fecal flora for use in the amelioration, stabilization,treatment and/or prevention of an infection, disease, treatment,poisoning or a condition having a bowel dysfunction component orside-effect, or for the amelioration, treatment and/or prevention of aconstipation, for the treatment of an abdominal pain, a non-specificabdominal pain or a diarrhea, a diarrhea caused by: a drug side effector a psychological condition or Crohn's Disease, a poison, a toxin or aninfection, a toxin-mediated traveler's diarrhea, or a Clostridium or aC. perfringens welchii or a C. difficile infection or apseudo-membranous colitis associated with a Clostridium infection.

According to an eleventh aspect of the present invention, there isprovided use of an entire (or substantially entire) microbiota, atreated or untreated fecal flora sample, a complete or partial fecalflora sample, a fecal flora substantially or completely purified ofnon-fecal floral fecal material, or a partially, substantially orcompletely isolated or purified fecal flora in the preparation of amedicament for the amelioration, stabilization, treatment and/orprevention of an infection, disease, treatment, poisoning or a conditionhaving a bowel dysfunction component or side-effect, or for theamelioration, treatment and/or prevention of a constipation, for thetreatment of an abdominal pain, a non-specific abdominal pain or adiarrhea, a diarrhea caused by: a drug side effect or a psychologicalcondition or Crohn's Disease, a poison, a toxin or an infection, atoxin-mediated traveler's diarrhea, or a Clostridium or a C. perfringenswelchii or a C. difficile infection or a pseudo-membranous colitisassociated with a Clostridium infection.

According to a twelfth aspect of the present invention, there isprovided use of a device of the seventh aspect for delivering a fecalmaterial or an entire (or substantially entire) microbiota, a treated oruntreated fecal flora sample, a complete or partial fecal flora sample,a fecal flora substantially or completely purified of non-fecal floralfecal material, or a partially, substantially or completely isolated orpurified fecal flora in the amelioration, stabilization, treatmentand/or prevention of an infection, disease, treatment, poisoning or acondition having a bowel dysfunction component or side-effect, or forthe amelioration, treatment and/or prevention of a constipation, for thetreatment of an abdominal pain, a non-specific abdominal pain or adiarrhea, a diarrhea caused by: a drug side effect or a psychologicalcondition or Crohn's Disease, a poison, a toxin or an infection, atoxin-mediated traveler's diarrhea, or a Clostridium or a C. perfringenswelchii or a C. difficile infection or a pseudo-membranous colitisassociated with a Clostridium infection.

In alternative embodiments, the invention provides compositions(including formulations, pharmaceutical compositions, foods, feeds,supplements, products of manufacture, and the like) comprising: deliveryvehicle, formulation, container or device, comprising a treated oruntreated fecal flora, or a partially, substantially or completelyisolated fecal flora; and methods of making and using them.

In alternative embodiments, the invention provides delivery vehicles,formulations, pharmaceutical preparations, products of manufacture,containers or devices, comprising: a treated or untreated fecal flora,an entire (or substantially entire) microbiota, and/or a partially,substantially or completely isolated fecal flora, made by a processcomprising:

(a) (i) providing a treated or untreated fecal sample, or a samplecomprising an entire (or substantially entire) microbiota, or acomposition comprising a complete or partial fecal flora, or apartially, substantially or completely isolated or purified fecal flora;and, a delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device, and

-   -   (ii) placing the treated or untreated fecal sample, the        partially, substantially or so completely isolated or purified        fecal flora, the entire (or substantially entire) microbiota, or        a composition comprising a complete or partial fecal flora or        partially, substantially or completely isolated or purified        fecal flora, in the delivery vehicle, formulation,        pharmaceutical preparation, product of manufacture, container or        device, and

optionally creating a substantially or completely oxygen-freeenvironment in the delivery vehicle, formulation, pharmaceuticalpreparation, product of manufacture, container or device, and

optionally the delivery vehicle, formulation, pharmaceuticalpreparation, product of manufacture, container or device is sterile orbacteria-free before the placing of the treated or untreated fecalsample, or the partially, substantially or completely isolated orpurified fecal flora;

(b) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of (a), wherein the deliveryvehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device is made substantially or completelyoxygen free (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% oxygen free) by:incorporating into the delivery vehicle, formulation, pharmaceuticalpreparation, product of manufacture, container or device a built in orclipped-on oxygen-scavenging mechanism; and/or, the delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device comprises or is coated with an oxygen scavengingmaterial; and/or completely or substantially replacing the air in thedelivery vehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device with nitrogen and/or other inertnon-reactive gas or gases;

(c) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of (a) or (b), wherein thedelivery vehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device simulates (creates) partially,substantially or completely an anaerobic environment;

(d) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (c),wherein the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device is manufactured, labelled orformulated for human or animal use;

(e) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of (d), wherein the animaluse is for a veterinary use;

(f) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (e),wherein a stabilizing agent or a glycerol is added to, or mixed into,the treated or untreated fecal sample, entire (or substantially entire)microbiota, or partially, substantially or completely isolated fecalflora, before storage or freezing, spray-drying, freeze-drying orlyophilizing;

(g) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (f),wherein the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device is initially manufactured orformulated as a liquid, a suspension, a gel, a geltab, a semisolid, atablet, a sachet, a lozenge or a capsule, or as an enteral formulation,or re-formulated for final delivery as a liquid, a suspension, a gel, ageltab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or asan enteral formulation;

(h) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (g),wherein the fecal sample is treated such that the fecal flora isseparated from rough particulate matter in the fecal sample by:homogenizing, centrifuging and/or filtering a rough particulate matteror a non-floral matter of the fecal material, or by plasmapheresis,centrifugation, celltrifuge, column chromatography (e.g., affinitychromatography), immunoprecipitation (e.g., antibodies fixed to a solidsurface, such as beads or a plate);

(i) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (h),wherein the treated or untreated fecal flora, entire (or substantiallyentire) microbiota, or partially, substantially or completely isolatedor purified fecal flora, is lyophilized, freeze-dried or frozen, orprocessed into a powder;

(j) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (i),wherein the fecal flora (including e.g., the entire (or substantiallyentire) microbiota) is initially derived from an individual screened ortested for a disease or infection, and/or the fecal flora is initiallyderived from an individual screened to have a normal, healthy or wildtype population of fecal flora;

(k) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (j),wherein a substantially isolated or a purified fecal flora or entire (orsubstantially entire) microbiota is (comprises) an isolate of fecalflora that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%/isolated or pure, orhaving no more than about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%,0.8%, 0.9% or 1.0% or more non-fecal floral material; or

(l) the delivery vehicle, formulation, pharmaceutical preparation,product of manufacture, container or device of any of (a) to (j),wherein the amount of the treated or untreated fecal sample, entire (orsubstantially entire) microbiota, or the partially, substantially orcompletely isolated or purified fecal flora is formulated for orcalibrated for repeat or multiple delivery or infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, the delivery vehicle, formulation,pharmaceutical preparation, product of manufacture, container or deviceof the invention further comprises:

a saline, a media, a defoaming agent, a surfactant agent, a lubricant,an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, acontrast agent, a dispersal agent, a buffer or a buffering agent, asweetening agent, a debittering agent, a flavouring agent, a pHstabilizer, an acidifying agent, a preservative, a desweetening agentand/or colouring agent;

at least one vitamin, mineral and/or dietary supplement, whereinoptionally the vitamin comprises a thiamine, riboflavin, nicotinic acid,pantothenic acid, pyridoxine, biotin, folic acid, vitamin B₁₂, lipoicacid, ascorbic acid, vitamin A, vitamin D, vitamin E, vitamin K, acholine, a camitine, and/or an alpha, beta and/or gamma carotene; or

a prebiotic nutrient, wherein optionally the prebiotic comprises anyingredient that stimulates the stability, growth and/or activity of thefecal flora or fecal bacteria, or optionally comprises polyols,fructooligosaccharides (FOSs), oligofructoses, inulins,galactooligosaccharides (GOSs), xylooligosaccharides (XOSs),polydextroses, monosaccharides, tagatose, and/or mannooligosaccharides.

In alternative embodiments, the invention provides products (articles)of manufacture comprising a delivery vehicle, formulation,pharmaceutical preparation, container or device of the invention.

In alternative embodiments, the invention provides methods for making adelivery vehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device, comprising a treated or untreatedfecal flora, entire (or substantially entire) microbiota, or apartially, substantially or completely isolated or purified fecal flora,comprising:

(a) (i) providing a treated or untreated fecal sample, or a compositioncomprising a complete or partial fecal flora, an entire (orsubstantially entire) microbiota, or a partially, substantially orcompletely isolated or purified fecal flora; and, a delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device, and

-   -   (ii) placing the treated or untreated fecal sample, the        partially, substantially or completely isolated or purified        fecal flora, the entire (or substantially entire) microbiota, or        a composition comprising a complete or partial fecal flora or        partially, substantially or completely isolated or purified        fecal flora, in the delivery vehicle, formulation,        pharmaceutical preparation, product of manufacture, container or        device, and creating a substantially or completely oxygen-free        environment in the container or device;

(b) the method of (a), wherein the delivery vehicle, formulation,pharmaceutical preparation, product of manufacture, container or deviceis made substantially or completely oxygen free by: incorporating intothe delivery vehicle, formulation, container or device a built in orclipped-on oxygen-scavenging mechanism; and/or, the delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device comprises or is coated with an oxygen scavengingmaterial; and/or completely or substantially replacing the air in thedelivery vehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device with nitrogen and/or other inertnon-reactive gas or gases;

(c) the method of (a) or (b), wherein the delivery vehicle, formulation,pharmaceutical preparation, product of manufacture, container or devicesimulates (creates) partially, substantially or completely an anaerobicenvironment;

(d) the method of any of (a) to (c), wherein the delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device is manufactured, labelled or formulated for human oranimal use;

(e) the method of (d), wherein the animal use is for a veterinary use;

(f) the method of any of (a) to (e), wherein a prebiotic, a stabilizingagent or a glycerol is added to, or mixed into, the treated or untreatedfecal sample, or partially, substantially or completely isolated orpurified fecal flora, before storage or freeze-drying, spray-drying,freezing or lyophilizing;

(g) the method of any of (a) to (f), wherein the delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device is initially manufactured or formulated as a liquid,a suspension, a gel, a geltab, a semisolid, a tablet, a sachet, alozenge or a capsule, or as an enteral formulation, or re-formulated forfinal delivery as a liquid, a suspension, a gel, a geltab, a semisolid,a tablet, a sachet, a lozenge or a capsule, or as an enteralformulation;

(h) the method of any of (a) to (g), wherein the fecal sample is treatedsuch that the fecal flora is separated from rough particulate matter inthe fecal sample by: homogenizing, centrifuging and/or filtering a roughparticulate matter or a non-floral matter of the fecal material, or byplasmapheresis, centrifugation, celltrifuge, column chromatography(e.g., affinity chromatography), immunoprecipitation (e.g., antibodiesfixed to a solid surface, such as beads or a plate);

(i) the method of any of (a) to (h), wherein the treated or untreatedfecal flora, or partially, substantially or completely isolated orpurified fecal flora, is lyophilized, freeze-dried or frozen, orprocessed into a powder;

(j) the method of any of (a) to (i), wherein the fecal flora isinitially derived from an individual screened or tested for a disease orinfection, and/or the fecal flora is initially derived from anindividual screened to have a normal, healthy or wild type population offecal flora; or (k) the method of any of (a) to (j), further comprisingadding to the treated or untreated fecal flora, or adding to a liquid orsolution used to isolate or purify, store, freeze, freeze-dry,spray-dry, lyophilize, transport, reconstitute and/or deliver a treatedor untreated fecal flora (optionally an entire (or substantially entire)microbiota, a partially, substantially or completely isolated orpurified fecal flora, or a composition comprising a fecal florasubstantially or completely purified of non-fecal floral fecal materialof the invention):

a saline, a media, a defoaming agent, a surfactant agent, a lubricant,an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, acontrast agent, a dispersal agent, a buffer or a buffering agent, asweetening agent, a debittering agent, a flavouring agent, a pHstabilizer, an acidifying agent, a preservative, a desweetening agentand/or colouring agent, and/or

at least one vitamin, mineral and/or dietary supplement, whereinoptionally the vitamin comprises a thiamine, riboflavin, nicotinic acid,pantothenic acid, pyridoxine, biotin, folic acid, vitamin B₁₂, lipoicacid, ascorbic acid, vitamin A, vitamin D, vitamin E, vitamin K, acholine, a camitine, and/or an alpha, beta and/or gamma carotene, and/or

a prebiotic nutrient, wherein optionally the prebiotic comprises anyingredient that stimulates the stability, growth and/or activity of thefecal flora or fecal bacteria, or optionally comprises polyols,fructooligosaccharides (FOSs), oligofructoses, inulins,galactooligosaccharides (GOSs), xylooligosaccharides (XOSs),polydextroses, monosaccharides, tagatose, and/or mannooligosaccharides;

(l) the method of any of (a) to (k), wherein an entire (or substantiallyentire) microbiota, a substantially isolated or a purified fecal florais (comprises) an isolate of fecal flora that is at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%or 99.9% isolated or pure, or having no more than about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% or more non-fecalfloral material; or

(m) the method of any of (a) to (l), wherein the amount of the entire(or substantially entire) microbiota, the treated or untreated fecalsample, or the partially, substantially or completely isolated orpurified fecal flora is formulated for or calibrated for repeat ormultiple delivery or infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, the invention provides methods for theamelioration, stabilization, treatment and/or prevention of aninfection, disease, treatment, poisoning or a condition having a boweldysfunction component or side-effect, or for the amelioration,stabilization, treatment and/or prevention of a constipation, for thetreatment of an abdominal pain, a non-specific abdominal pain or adiarrhea, a diarrhea caused by: a drug side effect or a psychologicalcondition or Crohn's Disease, a poison, a toxin or an infection, atoxin-mediated traveler's diarrhea, or a Clostridium or a C. difficileor a pseudo-membranous colitis associated with a Clostridium infection,comprising administering to an individual in need thereof a deliveryvehicle, formulation, pharmaceutical preparation, product ofmanufacture, container or device of the invention, or a product(article) of manufacture of the invention.

In alternative embodiments, the invention provides methods for theamelioration, stabilization, treatment and/or prevention of aninfection, disease, treatment, poisoning or a condition having a boweldysfunction component or side-effect comprising administering to anindividual in need thereof a delivery vehicle, formulation,pharmaceutical preparation, product of manufacture, container or deviceof the invention, or a product (article) of manufacture of theinvention, or their contents (e.g., the bacterial flora containedtherein).

In alternative embodiments, the amount of the treated or untreated fecalsample, or the partially, substantially or completely isolated orpurified fecal flora is formulated for or calibrated for repeat ormultiple delivery or infusions; or the partially, substantially orcompletely isolated or purified fecal flora is delivered in repeated ormultiple infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, of the methods the infection, disease,treatment, poisoning or condition having a bowel dysfunction componentor side-effect comprises an inflammatory bowel disease (IBD), Crohn'sdisease, hepatic encephalopathy, enteritis, colitis, Irritable BowelSyndrome (IBS), fibromyalgia (FM), chronic fatigue syndrome (CFS),depression, attention deficit/hyperactivity disorder (ADHD), multiplesclerosis (MS), systemic lupus erythematosus (SLE), travelers' diarrhea,small intestinal bacterial overgrowth, chronic pancreatitis, apancreatic insufficiency, exposure to a poison or a toxin or for aninfection, a toxin-mediated traveler's diarrhea, a poisoning, apseudomembranous colitis, a Clostridium infection, a C. perfringenswelchii or a Clostridium difficile infection, a neurological condition,Parkinson's disease, myoclonus dystonia, autism, amyotrophic lateralsclerosis, multiple sclerosis, Grand mal seizures or petit mal seizures.In alternative embodiments, the amount of the treated or untreated fecalsample, or the partially, substantially or completely isolated orpurified fecal flora, is formulated for or calibrated for repeat ormultiple delivery or infusions; or the treated or untreated fecal sampleor the partially, substantially or completely isolated or purified fecalflora is delivered in repeated or multiple infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, the invention provides delivery vehicles,formulations, pharmaceutical preparations, products of manufacture,containers or devices comprising:

(a) an entire (or substantially entire) microbiota, a partially,substantially or completely isolated or purified fecal flora, or acomposition comprising a fecal flora substantially or completelypurified of non-fecal floral fecal material, and optionally furthercomprising an excipient, or a fluid, a saline, a buffer, a bufferingagent or a media, or a fluid-glucose-cellobiose agar (RGCA) media;

(b) the composition, container or device, formulation, or product ofmanufacture of (a), wherein the entire (or substantially entire)microbiota or the fecal flora is isolated or purified from a human or ananimal fecal material;

(c) the composition, container or device, formulation, or product ofmanufacture of (a) or (b), wherein the entire (or substantially entire)microbiota or the fecal flora is isolated or purified using a method orprotocol comprising use of a centrifuge, a celltrifuge, a column or animmuno-affinity column, or wherein the entire (or substantially entire)microbiota or the fecal flora is isolated or purified by a methodcomprising homogenizing, centrifuging and/or filtering a roughparticulate matter or a non-floral matter of the fecal material, or byplasmapheresis, centrifugation, celltrifuge, column chromatography(e.g., affinity chromatography), immunoprecipitation (e.g., antibodiesfixed to a solid surface, such as beads or a plate);

(d) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (c), wherein the entire (or substantiallyentire) microbiota or the substantially or completely isolated orpurified fecal flora, or the fecal flora substantially or completelypurified of non-fecal floral fecal material, is in a substantially orcompletely oxygen-free environment in the composition, container ordevice, formulation, or product of manufacture;

(e) the delivery vehicle, formulation, container or device of (d),wherein the composition, container or device, formulation, or product ofmanufacture is made substantially or completely oxygen free by:incorporating into the delivery vehicle, formulation, container ordevice a built in or clipped-on oxygen-scavenging mechanism; and/or, thedelivery vehicle, formulation, container or device comprises or iscoated with an oxygen scavenging material; and/or completely orsubstantially replacing the air in the delivery vehicle, formulation,container or device with nitrogen and/or other inert non-reactive gas orgases;

(f) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (c), wherein the entire (or substantiallyentire) microbiota, the substantially or completely isolated or purifiedfecal flora, or the fecal flora substantially or completely purified ofnon-fecal floral fecal material, is in a substantially or completelyanaerobic environment;

(g) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (f), wherein the delivery vehicle,formulation, container or device is manufactured, labelled or formulatedfor human or animal use;

(h) the composition, container or device, formulation, or product ofmanufacture of (g), wherein the animal use is for a veterinary use;

(i) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (h), wherein a stabilizing agent or aglycerol is added to, or mixed into, the entire (or substantiallyentire) microbiota, or the partially, substantially or completelyisolated or purified fecal flora, or the composition comprising a fecalflora substantially or completely purified of non-fecal floral fecalmaterial, before storage or freezing, freeze-drying, spray-drying orlyophilizing;

(j) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (f), wherein the composition, container ordevice, formulation, or product of manufacture is initially manufacturedor formulated as a liquid, a suspension, a gel, a geltab, a semisolid, atablet, a sachet, a lozenge or a capsule, or as an enteral formulation,or re-formulated for final delivery as a liquid, a suspension, a gel, ageltab, a semisolid, a tablet, a sachet, a lozenge or a capsule, or asan enteral formulation;

(k) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (j), wherein the entire (or substantiallyentire) microbiota, or the partially, substantially or completelyisolated or purified fecal flora, or the composition comprising a fecalflora substantially or completely purified of non-fecal floral fecalmaterial, is lyophilized, freeze-dried, in powder form, or frozen;

(l) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (k), wherein the entire (or substantiallyentire) microbiota or the fecal flora is initially derived from anindividual screened or tested for a disease or infection, and/or theentire (or substantially entire) microbiota or the fecal flora isinitially derived from an individual screened to have a normal, healthyor wild type population of fecal flora; or

(m) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (l), further comprising adding to theentire (or substantially entire) microbiota, the partially,substantially or completely isolated or purified fecal flora, or acomposition comprising a fecal flora substantially or completelypurified of non-fecal floral fecal material, or adding to a liquid orsolution used to isolate or purity, store, freeze, freeze-dry,spray-dry, lyophilize, transport, reconstitute and/or deliver a treatedor untreated fecal flora:

a saline, a media, a defoaming agent, a surfactant agent, a lubricant,an acid neutralizer, a marker, a cell marker, a drug, an antibiotic, acontrast agent, a dispersal agent, a buffer or a buffering agent, asweetening agent, a debittering agent, a flavouring agent, a pHstabilizer, an acidifying agent, a preservative, a desweetening agentand/or colouring agent, and/or

at least one vitamin, mineral and/or dietary supplement, whereinoptionally the vitamin comprises a thiamine, riboflavin, nicotinic acid,pantothenic acid, pyridoxine, biotin, folic acid, vitamin B₁₂, lipoicacid, ascorbic acid, vitamin A, vitamin D, vitamin E, vitamin K, acholine, a camitine, and/or an alpha, beta and/or gamma carotene, and/or

a prebiotic nutrient, wherein optionally the prebiotic comprises anyingredient that stimulates the stability, growth and/or activity of theentire (or substantially entire) microbiota or the fecal flora or fecalbacteria, or optionally comprises polyols, fructooligosaccharides(FOSs), oligofructoses, inulins, galactooligosaccharides (GOSs),xylooligosaccharides (XOSs), polydextroses, monosaccharides, tagatose,and/or mannooligosaccharides;

(n) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (m), wherein a substantially isolated or apurified fecal flora is (comprises) an isolate of the entire (orsubstantially entire) microbiota or fecal flora that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%,99.8% or 99.9% isolated or pure, or having no more than about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% or more non-fecalfloral material; or

(m) the composition, container or device, formulation, or product ofmanufacture of any of (a) to (l), wherein the amount of the entire (orsubstantially entire) microbiota or the treated or untreated fecalsample, or the partially, substantially or completely isolated orpurified fecal flora is formulated for or calibrated for repeat ormultiple delivery or infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, the invention provides methods for theamelioration, stabilization, treatment and/or prevention of aninfection, disease, treatment, poisoning or a condition having a boweldysfunction component or side-effect comprising administering to anindividual in need thereof a composition, container or device,formulation, or product of manufacture of the invention.

In alternative embodiments, of the methods the infection, disease,treatment, poisoning or condition having a bowel dysfunction componentor side-effect comprises an inflammatory bowel disease (IED), Crohn'sdisease, hepatic encephalopathy, enteritis, colitis, irritable bowelsyndrome (IES), fibromyalgia (FM), chronic fatigue syndrome (CFS),depression, attention deficit/hyperactivity disorder (ADHD), multiplesclerosis (MS), systemic lupus erythematosus (SLE), travelers' diarrhea,small intestinal bacterial overgrowth, chronic pancreatitis, apancreatic insufficiency, exposure to a poison or a toxin or for aninfection, a toxin-mediated traveler's diarrhea, a poisoning, apseudomembranous colitis, a Clostridium infection, a C. perfringenswelchii or a Clostridium difficile infection, a neurological condition,Parkinson's disease, myoclonus dystonia, autism, amyotrophic lateralsclerosis, multiple sclerosis, Grand mal seizures or petit mal seizures.

In alternative embodiments, the invention provides methods for theamelioration, stabilization, treatment and/or prevention of, ordecreasing or delaying the symptoms of, an infection, disease,treatment, poisoning or a condition having a bowel dysfunction componentor side-effect, or for the amelioration, treatment and/or prevention ofa constipation, for the treatment of an abdominal pain, a non-specificabdominal pain or a diarrhea, a diarrhea caused by: a drug side effector a psychological condition or Crohn's Disease, a poison, a toxin or aninfection, a toxin-mediated traveler's diarrhea, or a Clostridium or aC. perfringens welchii or a C. difficile infection or apseudo-membranous colitis associated with a Clostridium infection, orfor preventing, decreasing or delaying the symptoms of, amelioratingstabilizing, or treating individuals (e.g., patients or animals) withspondyloarthropathy, spondylarthritis or sacroileitis (an inflammationof one or both sacroiliac joints); a nephritis syndrome; an inflammatoryor an autoimmune condition having a gut or an intestinal component suchas lupus, irritable bowel syndrome (IBS or spastic colon) or a colitissuch as Ulcerative Colitis or Crohn's Colitis; constipation, autism;degenerative neurological diseases such as amyotrophic lateral sclerosis(ALS), Multiple Sclerosis (MS) or Parkinson's Disease (PD); a MyoclonusDystonia (e.g., Steinert's disease or proximal myotonic myopathy); anautoimmune disease such as Rheumatoid Arthritis (RA) or juvenileidiopathic arthritis (JIA); Chronic Fatigue Syndrome (including benignmyalgic encephalomyelitis, chronic fatigue immune dysfunction syndrome,chronic infectious mononucleosis, epidemic myalgic encephalomyelitis);obesity; hypoglycemia, pre-diabetic syndrome, type I diabetes or type IIdiabetes; Idiopathic thrombocytopenic purpura (ITP); an acute or chronicallergic reaction such as hives, a rash, a urticaria or a chronicurticaria; and/or insomnia or chronic insomnia, Grand mal seizures orpetit mal seizures, comprising:

administering to an individual in need thereof a delivery vehicle,formulation, pharmaceutical preparation, product of manufacture,container or device of the invention, or a product (article) ofmanufacture of the Invention, in single, repeat or multipleadministrations, deliveries or infusions.

In alternative embodiments, the amount of the entire (or substantiallyentire) microbiota, the treated or untreated fecal sample, or thepartially, substantially or completely isolated or purified fecal flora,is formulated for or calibrated for repeat or multiple delivery orinfusions; or the entire (or substantially entire) microbiota, thetreated or untreated fecal sample or the partially, substantially orcompletely isolated or purified fecal flora is delivered in repeated ormultiple infusions,

wherein optionally the repeated or multiple administration, delivery,infusion or implantation protocol comprises infusions done daily for thefirst about 10 days, second daily for about 10 days, third daily thenfourth daily possibly weekly and then optionally maintain second or moreweekly infusions until the histology reverses towards normality.

In alternative embodiments, the invention provides devices fordelivering a (bacterial flora-comprising) composition of the invention,or an entire (or substantially entire) microbiota or a fecal materialcomprising:

(a) a device as illustrated in FIG. 1 or FIG. 2, or equivalent thereof;or

(b) (1) a bag or container comprising an exit aperture operablyconnected to the proximal end of a flexible tube or equivalent,

wherein the bag or container is optionally made of a material imperviousto a gas or to oxygen,

and optionally the bag or container is made of a flexible material, or apolyethylene terephthalate polyester film-comprising (or a MYLAR™comprising) material,

and optionally the bag or container is an (IV-like) intravenous-likebag,

and optionally the bag or container has an attachment that will allowthe bag to be hung on a stand, e.g., to be positioned/hung above anendoscope,

and optionally the bag or container is operably connected via an open orclose valve or equivalent to a negative pressure device that can removeall gas or air from the bag,

and optionally the bag or container is operably connected via an open orclose valve or equivalent to a fluid source or storage container forflushing out the bag through the exit aperture,

and optionally the fluid source or storage container is under positivepressure, and optionally the flexible tube or equivalent comprises atleast one clip or close valve or one way valve to prevent backwash ofmaterial from distal to proximal portions of the tube, or from the tubeback to the bag or container;

(2) an open or close valve or equivalent or an obdurator screwtop at thedistal end of the flexible tube or equivalent,

and optionally a Luer lock tip for attachment to a colonoscope or anendoscopic Luer lock port or equivalent, wherein optionally the Luerlock tip is built into the valve, or is separate from the valve,

and optionally an enema tube tip for attachment to an enema tube ordevice or equivalent,

wherein optionally the enema tube tip is built into the valve, or isseparate from the valve, and optionally further comprising a safetydevice or safety clip to close the distal aperture in case the valve orLuer lock tip, or enema tip, is lost (flies off) under pressure; and

(3) a pump, or a hand pump, for moving material in the bag or containerthrough the flexible tube or equivalent and out the distal end or outthe open or close valve or equivalent; or

(c) the device of (a) or (b), further comprising a fecal material or acomposition of the invention.

In alternative embodiments, the invention provides bags or containerscomprising an entire (or substantially entire) microbiota, or a treatedor untreated fecal flora, or a partially, substantially or completelyisolated fecal flora, or a composition of the invention (e.g., aformulation comprising an entire (or substantially entire) microbiota, apartially, substantially or completely isolated or purified fecal flora,or a composition comprising a fecal flora substantially or completelypurified of non-fecal floral fecal material), and optionally furthercomprising an excipient, or a fluid, a saline, a buffer, a bufferingagent or a media, or a fluid-glucose-cellobiose agar (RGCA) media,wherein the bag or container is structurally the same as or similar to abag or container of a device of the invention (e.g., as illustrated inFIG. 1 or FIG. 2), wherein optionally the interior of the bag issubstantially or completely an oxygen-free environment, or the interiorof the bag is substantially or completely similar to an anaerobicenvironment.

In alternative embodiments, specific anti C. difficile oral antibodies(for example avian) can be added to a solution (e.g., a saline, media,buffer) used to isolate or purify, store, freeze, freeze-dry, spray-drylyophilize, transport, reconstitute and/or deliver a composition (e.g.,a partially, substantially or completely isolated or purified fecalflora, or a composition comprising a fecal flora substantially orcompletely purified of non-fecal floral fecal material) of theinvention. The combination of the product with these specific anti C.difficile oral antibodies enhances the eradication mechanism of theproduct.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

FIG. 1A illustrates an exemplary storage device of the invention; and,FIG. 1B illustrates an exemplary delivery device of the invention, asdescribed below.

FIG. 2 illustrates an exemplary delivery device of the invention.

Like reference symbols in the various drawings indicate like elements.Reference will now be made in detail to various exemplary embodiments ofthe invention, examples of which are illustrated in the accompanyingdrawings. The following detailed description is provided to give thereader a better understanding of certain details of aspects andembodiments of the invention, and should not be interpreted as alimitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, the invention provides compositions, e.g.,formulations and pharmaceutical preparations, products of manufacture,and containers and delivery vehicles, and devices and deliverymaterials, comprising treated and/or isolated faecal (fecal) materialfor faecal floral transplantation. In one embodiment, the treated and/orisolated fecal material of the invention comprising faecal floral (e.g.,bacteria) is transplanted between different individuals, e.g., human tohuman or between animals. In one embodiment, the treated fecal materialof the invention is transplanted back into the same individual fromwhich it was collected, e.g., to repopulate a colon after drug treatment(e.g., antibiotic treatment or chemotherapy) or after an orthostaticlavage, e.g., for inducing the purgation (e.g., cleansing) of agastrointestinal (GI) tract, including a colon.

The invention provides methods for the amelioration, stabilization, ortreatment of a bowel disease or infection comprising use of a deliveryvehicle, formulation, product of manufacture, or container or device ofthe invention; e.g., as a fecal bacteriotherapy, fecal transfusion,fecal transplant, or human probiotic infusion (HPI). In alternativeembodiments, the invention provides methods for ameliorating,stabilizing, treating or preventing any infection, bowel disease or tocondition having a bowel dysfunction component, for example, apoisoning, a pseudomembranous colitis, a Clostridium difficileinfection, an inflammatory bowel disease (IBD), Crohn's disease, hepaticencephalopathy, enteritis, colitis, irritable bowel syndrome (IBS),fibromyalgia (FM), chronic fatigue syndrome (CFS), depression, attentiondeficit/hyperactivity disorder (ADHD), multiple sclerosis (MS), systemiclupus erythematosus (SLE), travelers' diarrhea, small intestinalbacterial overgrowth, chronic pancreatitis, or a pancreaticinsufficiency.

For example, in one embodiment, as antibiotics do not eradicate C.difficile and its spore, a delivery vehicle, formulation, product ofmanufacture, or container or device of the invention comprising treatedand/or isolated fecal flora can ameliorate, stabilize or eradicate C.difficile (or the pseudo-membranous colitis associated with thisinfection) when infused into a colon of the infected or ill individual,e.g., a patient or animal. In alternative embodiments the fecal floraobtained from a donor (which in treated or isolated form is inalternative embodiments in a delivery vehicle, formulation, product ofmanufacture, or container or device of the invention) comprises a partof, substantially all of, or all of the infected or ill recipient'smissing or inadequate (e.g., in numbers or function) fecal flora, e.g.,bacteria. While the invention is not limited by any particular mechanismof action, in some embodiments it is the transfer of the equivalent of:a part of, substantially all of, or all of the fecal flora of theinfected individual from the donor to the recipient (e.g., from human tohuman) that ameliorates or eradicates the infection or thepseudo-membranous colitis associated with this infection.

In alternative embodiments, the compositions, e.g., formulations andpharmaceutical preparations, and devices, delivery materials, deliveryvehicles, products of manufacture, containers and devices of theinvention allow the safe transplantation of fecal flora (e.g., humanflora) components to individuals in need thereof, e.g., to infected,sick and dying patients, thus providing a consistently safe yetfunctioning flora for delivery to a recipient or patient.

In alternative embodiments, the invention provides a reliable method forproducing standardized fresh fecal flora which can have a long shelflife. For example, in one embodiment, the delivery vehicle, formulation,pharmaceutical preparation, product of manufacture, container or devicecomprising the fecal flora comprises a substantially or completelyoxygen-free environment. In another embodiment, nutrients such as“prebiotic nutrients” can be added (e.g., in dry or liquid forms) to asolution (e.g., a saline, media, buffer) used to isolate or purify,store, freeze, freeze-dry, spray-dry, lyophilize, transport,reconstitute and/or deliver a composition (e.g., a partially,substantially or completely isolated or purified fecal flora, or acomposition comprising a fecal flora substantially or completelypurified of non-fecal floral fecal material) of the invention. Aprebiotic nutrient can be any ingredient that stimulates the stability,growth and/or activity of the fecal flora, e.g., bacteria; for example,in alternative embodiments, polyols, fructooligosaccharides (FOSs),oligofructoses, inulins, galactooligosaccharides (GOSs),xylooligosaccharides (XOSs), polydextroses, monosaccharides such astagatose, and/or mannooligosaccharides are used as prebiotics topractice this invention. In one embodiment, the prebiotics are added toprevent “shock” to the fecal flora subsequent to their isolation orpurification, freezing, freeze-drying, spray-drying, reconstitution insolution and the like.

In alternative embodiments, components of the compositions, e.g.,delivery vehicles, formulations and pharmaceutical preparations,products of manufacture, or containers or devices, of the inventioncomprise an entire (or substantially entire) microbiota, or aBacteroides and/or Firmicutes in large numbers (e.g., a largerproportion of Bacteroides and/or Firmicutes is present that is normallyfound in situ), e.g., to be able to ameliorate and/or eradicate a C.difficile infection and/or the pseudo-membranous colitis associated withthis infection. In alternative embodiments, the compositions, e.g.,delivery vehicles, formulations and pharmaceutical preparations,products of manufacture, or containers or devices, of the invention canbe available (e.g., formulated and/or dosaged for) for recurrent use inindividuals, e.g., in patients or animals, with the more difficult totreat conditions such as colitis (e.g., the pseudo-membranous colitis ofa C. difficile infection) and constipation.

In alternative embodiments, components of the compositions e.g.,delivery vehicles, formulations and pharmaceutical preparations,products of manufacture, or containers or devices, of the inventioncomprise a selection of bacterial species e.g. Bacteroides, Firmicutes,Bacillus thuringiensis (a bacterium capable of producing peptideantibiotics for C. difficile). The bacterial species may be separated bycelltrifugation or plasmapharesis.

In alternative embodiments the selection of bacterial species e.g.Bacteroides, Firmicutes, Bacillus thuringiensis may be added tocomponents of the compositions e.g., delivery vehicles, formulations andpharmaceutical preparations, products of manufacture, or containers ordevices as fortification of concentrations comprising the bacterialspecies to contain wild types of bacteria.

In alternative embodiments, compositions of the invention can beformulated as fecal slurries, saline or buffered suspensions (e.g., foran enema, suspended in a buffer or a saline), in a drink (e.g., a milk,yoghurt, a shake, a flavoured drink or equivalent) for oral delivery,and the like.

In alternative embodiments, compositions of the invention can beformulated as an enema product, a spray dried product, reconstitutedenema, a small capsule product, a small capsule product suitable foradministration to children, a bulb syringe, a bulb syringe suitable fora home enema with a saline addition, a powder product, a powder productin oxygen deprived sachets, a powder product in oxygen deprived sachetsthat can be added to, for example, a bulb syringe or enema, or a spraydried product in a device that can be attached to a container with anappropriate carrier medium such as yoghurt or milk and that can bedirectly incorporated and given as a dosing for example for children.

In one embodiment, compositions of the invention can be delivereddirectly in a carrier medium via a screw-top lid wherein the fecalmaterial is suspended in the lid and released on twisting the lidstraight into the carrier medium.

In alternative embodiments methods of delivery of compositions of theinvention include use of fecal slurries into the bowel, via an enemasuspended in saline or a buffer, orally in a drink (e.g., a milk,yoghurt, a flavoured drink and the like), via a small bowel infusion viaa nasoduodenal tube, via a gastrostomy, or by using a colonoscope. Insome embodiment, there may be advantages delivering via a colonoscope toinfuse as proximally as possible, and to detect any colonic pathology.

In alternative embodiments methods, fecal flora used in the compositionand methods of the invention is initially derived (entirely or in part)from an individual screened or tested for a disease or infection, and/orthe fecal flora is initially derived from an individual screened to havea normal, healthy or normal, representative “wild type” population offecal flora; e.g., a normal complement of a Bacteroides and/orFirmicutes, and/or other fecal flora such as Bacillus thuringiensis. Inone embodiment, depending on a deficiency of a floral (e.g., bacterial)specie or species in a donor fecal material, or to achieve a desiredeffect, one or more additional (or “supplemental”) species, e.g.,Bacteroides, Firmicutes and/or Bacillus thuringiensis species, is addedto (or is administered with) the delivered product either initially whenthe product is made, or at the time of delivery, e.g., the additionalspecies is/are mixed in before application to the individual (e.g.,patient or animal), e.g., when a powder, lyophilate, or freeze-driedcomposition is reconstituted for delivery; or the one or more additional(or “supplemental”) species can be co-administered. These additionalfloral species can be directly isolated or purified from a donor, or canbe expanded (cultured) for a time in vitro before addition, or can comefrom (be derived from) a pure culture, e.g., from an ATTC stock. Forexample, in some applications, e.g., to achieve a desired effect ortherapeutic outcome, a delivery of an enhanced amount of one or morefecal flora (e.g., bacterial) species is used, e.g., the deliveredproduct (e.g., an entire (or substantially entire) microbiota, or acomposition comprising a complete or partial fecal flora, or apartially, substantially or completely isolated or purified fecal flora)is enhanced with (is “spiked” with”) one or more additional (or“supplemental”) species, e.g., Bacteroides, Firmicutes and/or Bacillusthuringiensis species, which can be directly isolated from a donor, orcan come from a pure culture, and the like.

In some embodiments, selection of the donor is of crucial importance,e.g., to avoid infecting the recipient with a separate infection ordisease. In alternative embodiments the donor is tested (screened) atleast for e.g., retrovirus (e.g., human immunodeficiency virus, HIV);hepatitis A, B, and/or C; cytomegalovirus; Epstein-Barr virus,detectable parasites and/or bacterial pathogens, depending on the specieof the donor and recipient, e.g., human or animal.

In alternative embodiments, the invention provides a process forpreparing fecal flora (e.g., an entire (or substantially entire)microbiota) for transplantation, first comprising a collection from oneor more healthy (e.g., screened) donor(s). In alternative embodiments, afresh stool is transported via a stool collection device of theinvention, which in one embodiment comprises a suitably oxygen free (orsubstantially oxygen free) appropriate container. An exemplary suitablestool collection device 1 is shown in FIG. 1A. FIG. 1A shows anexemplary container of the invention for containing the stool andincluding a slot 2 for receiving the stool. The container may then beplaced into a bag 3 suitably a disposable leak proof ziplock/sealingbag.

In alternative embodiments, the container can be made oxygen free bye.g., incorporating into the container a built in or clipped-onoxygen-scavenging mechanism, e.g., oxygen scavenging pellets asdescribed e.g., in U.S. Pat. No. 7,541,091, In another embodiment, thecontainer itself is made of an oxygen scavenging material, e.g., oxygenscavenging iron, e.g., as described by O2BLOCK™, or equivalents, whichuses a purified and modified layered clay as a performance-enhancingcarrier of oxygen-scavenging iron; the active iron is dispersed directlyin the polymer. In one embodiment, oxygen-scavenging polymers are usedto make the container itself or to coat the container, or as pellets tobe added; e.g., as described in U.S. Pat. App. Pub. 20110045222,describing polymer blends having one or more unsaturated olefinichomopolymers or copolymers; one or more polyamide homopolymers orcopolymers; one or more polyethylene terephthalate homopolymers orcopolymers; that exhibit oxygen-scavenging activity. In one embodiment,oxygen-scavenging polymers are used to make the container itself or tocoat the container, or as pellets to be added; e.g., as described inU.S. Pat. App. Pub. 20110008554, describing compositions comprising apolyester, a copolyester ether and an oxidation catalyst, wherein thecopolyester ether comprises a polyether segment comprisingpoly(tetramethylene-co-alkylene ether). In one embodiment,oxygen-scavenging polymers are used to make the container itself or tocoat the container, or as pellets to be added; e.g., as described inU.S. Pat. App. Pub. 201000255231, describing a dispersed iron/saltparticle in a polymer matrix, and an oxygen scavenging film with oxygenscavenging particulates.

Alternatively, in addition to or in place of the oxygen-scavengingmechanism, the air in the container is replaced (completely orsubstantially) with nitrogen and/or other inert non-reactive gas orgases. In alternative embodiments, the container simulates (creates)partially, substantially or completely an anaerobic environment.

In alternative embodiments, the stool (e.g., fecal sample) is held in anaesthetically acceptable container that will not leak nor smell yetmaintain an anaerobic environment. In alternative embodiments, thecontainer is sterile before receiving the fecal flora.

In alternative embodiments, the container is maintained below roomtemperature, e.g., refrigerated, during most or all of its preparation,transportation and/or storage at e.g., a “stool bank” or at the sitewhere the transplantation will take place. For example, once deliveredto a “processing stool bank” it is stored in a cool room, cold containeror refrigerator to minimize flora metabolism. In alternativeembodiments, it is not to be frozen to prevent destruction of thebacterial cells of the stool.

In alternative embodiments, stabilizing agents such as glycerol areadded to the harvested and/or stored material. In one embodiment, thestool is frozen suddenly in liquid nitrogen or any similar coolant soe.g., it can be stored for prolonged periods of time while waitingprocessing.

In alternative embodiments, the stool is tested for various pathogens,as noted above. In alternative embodiments, once cleared of infectiveagents, it is homogenized and filtered to remove large particles ofmatter. In alternative embodiments, it is subdivided into desiredvolumes, e.g., which can be between 5 cc and 3 or more liters. Forexample, in one embodiment, a container comprises a 50 gram (g) stool,which can be held in an appropriate oxygen resistant plastic, e.g., ametallized polyethylene terephthalate polyester film, or a metallizedMYLAR™.

In alternative embodiments, as shown in FIG. 1B, the exemplarytherapeutic vehicle (delivery system) 10 and the equipment in which thestool material is held is an intravenous-like (IV-like) giving set 11,e.g., with a hand pump 12 attached to the set. Suitably the bag 11 ismetallised MYLAR™ which is impervious to gases. The hand pump 12 canallow the contents of the liquefied stool residing in the upper part ofthe plastic device to be easily pumped forward when the entire equipmenttubing is attached by Luer lock mechanism 13 to the colonoscope biopsychannel. In this way a colonoscope or even an enteroscope will becomethe delivery mechanism. For this embodiment, this would usually be intothe colon at any distance, and alternatively into the caecum. Inalternative embodiments, the material is passed into a terminal ileum oreven higher, as desired. In alternative embodiments, it can be infusedinto the duodenum or below with an enteroscope. In alternativeembodiments, C. difficile (or the pseudo-membranous colitis associatedwith this infection) is ameliorated or eradicated with the infused fecalsample, or treated stool.

Another alternative embodiment is shown in FIG. 2. In this embodimentthe therapeutic vehicle/delivery system 20 including an IV-like bag 21including saline (NaCl) 22 and stool/cells 23 of the invention. Inaddition to the hand pump 24 and Luer lock 25, the delivery system isprovided with a flushing port 26 (for flushing out the bag), a clip 27(to prevent backwash) and an enema tip 28 with Luer lock attachment.

In alternative embodiments, the transplant material is subject tohomogenization and straining. In alternative embodiments, this treatedmaterial Is placed into a container, e.g., a bag, that can be attachedto a nasogastric or naso-duodenal tube to allow the contents to beinfused e.g., into either a stomach, duodenum or the distal jejunum.Alternatively it can be kept in a container, e.g., a bag, which can beattached to an enema tip to be given as an enema.

In alternative embodiments, to separate the non-bacterial components andproduce a stable product that can be frozen or lyophilized and have along shelf life, the stool can be homogenized and filtered from roughparticulate matter. In alternative embodiments, the microscopicfiber/nonliving matter is then separated from the bacteria. Severalmethods can be used, including e.g., recurrent filtration with filtersizes, e.g., coming down to the size of the bacterium.

In one embodiment different filters are used to isolate the bacterialspp. This differs from the technique used for example by Williams in WO2011/033310A1 which uses a crude technique of filtration with a gauzeand is inferior to that of the present invention which utilisesdifferent sized filtration membranes to obtain the purified bacteria.

In one embodiment, a filtration procedure for filtering whole stool issuitably used to reach the highest concentration of almost 100%bacteria. In one embodiment, the filtering procedure is a two-stepprocedure suitably using glass fibre depth filters for initialclarification. In one embodiment, the stool is filtered under positivepressure. In one embodiment, this would be using a combination orsandwich configuration with a 30 micron PVDF filter. In one embodiment,this sandwich procedure will be filtering the product under positivepressure. Later, membrane concentration can, in one embodiment, be usedas another step to reduce the volume of the filtrate. In one embodiment,this can be done prior to freeze drying or spray drying under nitrogencover.

Alternative membranes that can be used for filtration include, but notlimited to, nylon filters, cellulose nitrate filters, PES filters,Teflon filters, mixed cellulose Ester filters, polycarbonate filters,polypropylene filters, PVC filters or quartz filters. Variouscombinations of these can be used to achieve a high purity of bacteriawith solids and liquid removed ready for freezing, spray-drying orlyophilisation.

For freezing, in alternative embodiments, the bacteria is held in aliquid that will prevent bursting of cells on thawing. This can includevarious stabilizers, e.g., glycerol and appropriate buffers, and/orethylene glycol. In alternative embodiments, cryo-protectance uses finalconcentrations of stabilizer(s) of between about 20% to 60%, dependingin the stabilizer(s) used; this helps stabilize proteins by preventingformation of ice crystals that would otherwise destroy proteinstructures.

In alternative embodiments, stabilizers that help reduce destruction ofliving bacteria include skim milk, erythritol, arabitol, sorbitol,glucose, fructose and other polyols. Polymers such as dextran andpolyethylene glycol can also be used to stabilize the faecal bacterialcells.

Mixing the appropriate amount of the bacterial flora with the stabilizerallows it to be snap frozen and kept frozen in the container that willbe used to transport it to appropriate facility where the patient willhave this infused after thawing.

In alternative embodiments, an entire (or substantially entire)microbiota, or an isolated and/or treated (e.g., purified or isolated)fecal material and/or flora, can be lyophilized or freeze dried, or theproduct can be frozen. In alternative embodiments freeze-drying allowsthe majority of cells to remain viable, and produces a powdered form ofthe product that can be gently pulverized into a powder. The powder, orlyophilized or freeze-dried flora or isolate, then can be encapsulatedinto a carrier, e.g., a tablet, geltab, pill or capsule, e.g., anenteric-coated capsule, or placed into oil-filled capsules foringestion. Alternatively, the freeze-dried or lyophilized product, orpowder, can be reconstituted before delivery to an individual in e.g., afluid, e.g., a sterile fluid, such as saline, a buffer or a media suchas a fluid-glucose-cellobiose agar (RGCA) media.

In alternative embodiments an entire (or substantially entire)microbiota, or an isolated and/or treated (e.g., purified or isolated)fecal material and/or flora can be spray-dried. In one embodimentspray-drying is preferred over freeze-drying or lyophilising.

In alternative embodiments, the entire (or substantially entire)microbiota, or isolated and/or treated fecal material and/or flora, issupplemented with wild type bacteria which has been derived from normalanimal (e.g., human) flora and/or recombinantly treated bacteria, e.g.,recombinant microorganisms that can synthesize a protein, small moleculeor carbohydrate that has a self-protective or ameliorative effect; orrecombinant microorganisms that can self-destruct when provided with anappropriate signal, e.g., a chemical delivered by ingestion.

In alternative embodiments, the transplantation product (e.g., acomposition of the invention comprising an isolated or purified fecalflora or an entire (or substantially entire) microbiota) is delivered byan infusion, e.g., through the rectum, stoma or down the uppergastrointestinal (GI) tract, or it can be used in a suppository pill,tablet or encapsulated form, e.g., with an enteric-coated graded releasecapsule or a tablet, e.g., with the addition of excipients. Inalternative embodiments the transplantation product is administered as asuppository to give the highest concentration in the rectum.

In one embodiment, the transplantation product (e.g., a composition ofthe invention comprising an isolated or purified fecal flora or anentire (or substantially entire) microbiota) is stored before, duringand/or after delivery to an individual, or for or during the delivery,in a fluid, e.g., a sterile fluid, such as saline, a buffer or a mediasuch as a fluid-glucose-cellobiose agar (RGCA) media.

In alternative embodiments, the compositions and methods of theinvention are used to ameliorate, stabilize, prevent and/or treat:various gastrointestinal conditions, e.g., C. difficile infection, C.perfringens welchii and other Clostridium infections, irritable bowelsyndrome, constipation, pouchitis, Crohn's disease and microscopiccolitis; neurological conditions such as Parkinson's disease, myoclonusdystonia, autism, amyotrophic lateral sclerosis and multiple sclerosis,Grand mal seizures or petit mal seizures. In one embodiment, theneurological conditions are treated by encapsulated or frozen material.In alternative embodiments, for colitis patients, recurrentadministration is required to suppress and reverse the inflammatorybowel disease and irritable bowel syndrome.

In alternative embodiments, a crude collected stool is filtered and/orhomogenized, and then its bacterial cells are separated (e.g., from the“crud” which contains the fiber) by plasmapheresis, centrifugation,celltrifuge, column chromatography (e.g., affinity chromatography),immunoprecipitation (e.g., antibodies fixed to a solid surface, such asbeads or a plate).

Centrifugation, including use of a “celltrifuge” (e.g., a Baxter modelMEDIFUGE 1215™) are processes that involve celltrifugal force toseparate mixtures. For “celltrifugation”, the densest components willthen fly to the outside of the spinning plates while the rest of thecomponents will migrate to the axis. The effect of the gravitationalforce will be increased by spinning the flattened product betweenrapidly moving glass plates. The centrifuge or celltrifuge can be set upsuch that the stool will be diluted adequately and set on a spinningcycle and collection of cells will occur only peripherally on thecentrifuge.

In alternative embodiments, wild type bacterial cells (including e.g.,an entire (or substantially entire) microbiota) separated or purifiede.g., by centrifugation, celltrifugation, plasmapheresis and F the like,are frozen using a cryoprotectant. In alternative embodiments, thismaterial is frozen in a container, e.g., a bag, which can then be usedto infuse through a colonoscope, naso-duodenal or nasogastric tube. Inalternative embodiments, it can be delivered to a facility (e.g., ahospital pharmacy) to be kept frozen, e.g., at −20° C. or below.Alternatively the centrifuged material can be lyophilized; and can beused either in a solution, gels, geltabs, pills, capsules or tablets, orsuppositories, e.g., to be reconstituted later as an enema or infuse setthrough a colonoscope.

In one embodiment the cryoprotectant is trehalose. Trehalose may alsofunction as a component upon reconstitution or as an additional agentprior to spray-drying or freeze-drying.

In alternative embodiments, solutions, gels, geltabs, pills, capsules ortablets comprising compositions of the invention (e.g., isolated orpurified fecal flora or an entire (or substantially entire) microbiota)can be taken long term, e.g., on a daily basis long term, e.g., for one,two, three or four weeks or months or more, to treat, stabilize,ameliorate or prevent a chronic and/or an immune condition such as e.g.,persistent infection, rheumatoid arthritis, systemic lupuserythematosus, autoimmune renal diseases, e.g., nephritis, severeobstruction, inflammatory bowel disease (IBD), irritable bowel syndrome(IBS), and other conditions set forth herein.

Preparations or Cultures of Entire Microbiota

in alternative embodiments, compositions (e.g., products of manufactureor formulations) of the invention, comprise preparations, formulations,cultures or culture extracts or isolates comprising an entire orsubstantially entire microbiota of an individual or specie, e.g., ahuman or other mammal. In alternative embodiments, the inventionprovides compositions and methods for preventing, decreasing thesymptoms of, ameliorating stabilizing, or treating various infections,disease or conditions comprising administration of these “entire orsubstantially entire microbiota” preparations (e.g., cultures or cultureisolates); for example, administering “entire or substantially entiremicrobiota” preparations for preventing, decreasing the symptoms of,ameliorating, stabilizing, or treating: spondyloarthropathy,spondylarthritis or sacrolileitis (an inflammation of one or bothsacroiliac joints); a nephritis syndrome; an inflammatory or anautoimmune condition having a gut or an intestinal component such aslupus, irritable bowel syndrome (IBS or spastic colon) or a colitis suchas Ulcerative Colitis or Crohn's Colitis; constipation, autism; adegenerative neurological diseases such as amyotrophic lateral sclerosis(ALS), Multiple Sclerosis (MS) or Parkinson's Disease (PD); a MyoclonusDystonia (e.g., Steinert's disease or proximal myotonic myopathy); anautoimmune disease such as Rheumatoid Arthritis (RA) or juvenileidiopathic arthritis (JIA); Chronic Fatigue Syndrome (including benignmyalgic encephalomyelitis, chronic fatigue immune dysfunction syndrome,chronic infectious mononucleosis, epidemic myalgic encephalomyelitis);obesity; hypoglycemia, pre-diabetic syndrome, type I diabetes or type IIdiabetes; Idiopathic thrombocytopenic purpura (ITP); an acute or chronicallergic reaction such as hives, a rash, a urticaria or a chronicurticaria; and/or insomnia or chronic insomnia, Grand mal seizures orpetit mal seizures.

In alternative embodiments, the invention provides compositions andmethods for administration of these “entire or substantially entiremicrobiota” preparations to prevent, decrease the symptoms of,ameliorate or treat various infections, diseases or conditionscomprising e.g., constipation, an inflammatory bowel disease (IBD),Crohn's disease, hepatic encephalopathy, enteritis, colitis, irritablebowel syndrome (IBS), fibromyalgia (FM), chronic fatigue syndrome (CFS),depression, attention deficit/hyperactivity disorder (ADHD), multiplesclerosis (MS), systemic lupus erythematosus (SLE), travelers' diarrhea,small intestinal bacterial overgrowth, chronic pancreatitis, apancreatic insufficiency, exposure to a poison or a toxin or for aninfection, a toxin-mediated traveler's diarrhea, a poisoning, apseudomembranous colitis, a Clostridium infection, a C. perfringenswelchii or a Clostridium difficile infection, a neurological condition,Parkinson's disease, myoclonus dystonia, autism, amyotrophic lateralsclerosis or multiple sclerosis, Grand mal seizures or petit malseizures.

While the invention is not limited by any particular mechanism ofaction, a treated or untreated fecal sample of the invention, or acomposition comprising a complete or partial fecal floral sample (e.g.,an entire or substantially entire microbiota”) of the invention, or apartially, substantially or completely isolated or purified fecal floraof the invention, when infused into a recipient (e.g., a human or amammal) colonize the gut. In one embodiment, these fecal floralpreparations are made (e.g., isolated) by filtering human flora, and/orby spinning or centrifuging, plasmapheresis, celltrifuge, columnchromatography (e.g., affinity chromatography), or immunoprecipitationand the like, to extract almost pure or substantially pure, or pure,fecal flora (e.g., “bacterial mass”).

In an alternative embodiment, compositions of the invention are preparedby culturing an entire (or substantially entire) microbiota culturedsimultaneously (e.g., all together without any pre-segregating out ofany particular bacterial species). In one embodiment, an “entire (orsubstantially entire) microbiota culture” sample is formulated e.g., asa liquid, or as a freeze dried or frozen product. In one embodiment,these preparations do not contain any (or are substantially free of)non-floral material, e.g., non-absorbed components normally present in afecal sample, e.g., a raw human stool. In one embodiment, a raw (e.g.,human) stool is made into a therapeutic agent or formulation.

In one embodiment, the invention provides methods of culturing an entiremammalian, e.g., a human, flora by taking a stool sample from a suitabledonor. In one embodiment, a suitable donor is a pathogen freeindividual; e.g., in one aspect a sample is collected from a donor whohas been classified as normal and free of any pathogens. In oneembodiment, as a stand-alone therapeutic or in conjunction with othertherapies, bacteria from lean donors may be used to treat obesity inobese patients.

In alternative embodiments, a culture is carried out for about 2, 3, 4,5, 6, 7 or 8 or more days under total or substantially total anaerobicconditions. Standard culturing procedures can be used using, e.g., anon-selective gut microbiota medium (GMM), and in one embodiment,incubated at (human) body temperature of about 36.8° C. An atmospheredevoid of (or substantially devoid of) oxygen and containing nitrogen,carbon dioxide and hydrogen can be used. Differing GMM can be used withvarying concentrations of the composition of the GMM.

Colonies or the cultured flora are then harvested by e.g., scraping witha sterile scraper. Harvested colonies or cultured flora can be frozene.g., in about minus 80° or below (e.g., in a freezer), using e.g., acryoprecipitate such as e.g., a glycerol, a cysteine or a milk. Suchcultures can then be aliquoted to be used only once (as re-culturing cancause a loss of adhesions). In one embodiment, methods can comprisere-culturing e.g., in a lipid culture medium resembling a GMM.

This entire medium can be frozen again using e.g., a glycerol with acysteine; and in one embodiment, can be kept frozen or freeze-dried.This can produce between about 108 to about 101° CFUs.

In alternative embodiments, powder, dried, frozen, freeze-dried orliquid or other forms of the cultured (e.g., human) bacteria (e.g., anentire or substantially entire microbiota”) can be formulated and/orused either as an enema, a food or food supplement or formulation (e.g.,added to a yoghurt, milk, drink, flavoured drink or a food), ordelivered as a capsule, tablet, geltab or the like (e.g., as an entericcoated capsule) to recolonise or alternatively or therapeutically“colonize” a gut flora.

In alternative embodiments cultured bacteria is added to the culture orsample or formulation of “entire (or substantially entire) microbiota”.For example, in one embodiment the first administration or the initialdaily formulations comprise only an “entire microbiota” formulation;while in other embodiments the first administration or the initial dailyformulations comprise both “entire microbiota” and additional culturedbacteria, e.g., cultured probiotic bacteria. In alternative embodiments,the less frequent formulations or dosages (which can be stepwise insmall or larger intervals, or periodic intervals, or intervals asdetermined by the physician or veterinarian according to rate ofimprovement, and the like) comprise only “entire microbiota”; while inother embodiments comprise both “entire microbiota” and the additionalcultured bacteria, e.g., cultured probiotic bacteria.

In alternative embodiments, to achieve a desired effect or therapeuticoutcome, the additional cultured bacteria (e.g., added to the “entiremicrobiota”) is a Bacteroides, Firmicutes and/or Bacillus thuringiensisspecies, which be directly isolated from a donor, or can come from apure culture, and the like. In alternative embodiments, a delivery of anenhanced amount of one or more fecal flora (e.g., bacterial) species isused, e.g., the delivered “entire microbiota” product is enhanced with(is “spiked” with”) one or more additional species, e.g., a Bacteroides,Firmicutes and/or Bacillus thuringiensis species.

Multiple or Repeated Infusions or Administrations

In alternative embodiments, compositions (e.g., products of manufactureor formulations) of the invention, including a treated or untreatedfecal sample, or a partially, substantially or completely isolated orpurified fecal flora, or a “culture of entire human microbiota” of theinvention, or an entire (or substantially entire) microbiota orcombination thereof is formulated for or calibrated for repeat ormultiple delivery or infusions. In alternative embodiments of methods ofthe invention, the partially, substantially or completely isolated orpurified fecal flora, e.g., of entire human microbiota, or an entire (orsubstantively entire) microbiota, or combination thereof, are deliveredor administered by repeat or multiple delivery or infusions.

The invention thus provides compositions and methods for treating,stabilizing, or ameliorating gut flora infections or conditions whichare difficult to permanently reverse, or for treating or amelioratingconditions characterised by gut flora infections which are difficult topermanently reverse. It has been discovered that multiple, repeatedinfusions can overcome gut flora infections or conditions that aredifficult to permanently reverse. In alternative embodiments, inpracticing the methods and/or compositions (e.g., products ofmanufacture or formulations) of the invention, multiple or repeatedfecal flora implantations (administrations, infusions) can overcome anunderlying tenacious ongoing flora infection in an individual (e.g., ananimal or a patient) with e.g., pathogenic and/or foreign bacterialstrains, or a chronic condition.

With inadequate elimination of the infective (e.g., pathogenic and/orforeign) bacteria, the ongoing original symptoms can return. It is knownthat bacteria sometimes do not divide and may live in biofilms in manywet (e.g., interior) surfaces of the body. Secondly, bacteria havespores which can be more difficult to eradicate at intermittent times ofsporulation. There are also dormant forms of bacteria that can be intra-and extra-cellular where they are much more difficult toeradicate—unless the dormant form is dividing. Finally, intracellularbacteria may wait until the gut wall cell in which they are housed isshed into the gut lumen re-infecting the flora. In alternativeembodiments, the multiple or repeated bowel flora infusions of themethods of the invention can, and may be required, to kill or otherwiseinactivate the viable (e.g., infective, pathogenic and/or foreign)bacteria which were protected inside the cell, biofilm and the like. Inalternative embodiments, the multiple or repeated bowel flora infusionsof the methods of the invention can, and may be required, to kill orotherwise inactivate bacterial cells that travel up crypts closer tolumen, where they are shed into the faecal stream and re-infect theindividual or patient.

Additionally, in alternative embodiments, the multiple (recurrent) orrepeated fecal flora implantations (administrations, infusions) ofmethods and/or compositions (e.g., products of manufacture orformulations) of the invention are effective for preventing,stabilizing, decreasing the symptoms of, ameliorating or treatingindividuals (e.g., patients) with: spondyloarthropathy, spondylarthritisor sacrolileltis (an inflammation of one or both sacroiliac joints); anephritis syndrome; an inflammatory or an autoimmune condition having agut or an intestinal component such as lupus, Irritable Bowel Syndrome(IBS or spastic colon) or a colitis such as Ulcerative Colitis orCrohn's Colitis; constipation, autism; a degenerative neurologicaldiseases such as amyotrophic lateral sclerosis (ALS), Multiple Sclerosis(MS) or Parkinson's Disease (PD); a Myoclonus Dystonia (e.g., Steinert'sdisease or proximal myotonic myopathy); an autoimmune disease such asRheumatoid Arthritis (RA) or juvenile idiopathic arthritis (JIA);Chronic Fatigue Syndrome (including benign myalgic encephalomyelitis,chronic fatigue immune dysfunction syndrome, chronic infectiousmononucleosis, epidemic myalgic encephalomyelitis); obesity;hypoglycemia, pre-diabetic syndrome, type I diabetes or type IIdiabetes; Idiopathic thrombocytopenic purpura (ITP); an acute or chronicallergic reaction such as hives, a rash, a urticaria or a chronicurticaria; and/or insomnia or chronic insomnia, Grand mal seizures orpetit mal seizures.

In alternative embodiments the invention is practiced (is carried out)either by use of methods or compositions of the invention, includingrecurrent enemas of human filtered stool, recurrent infusions through anaso-duodenal (ND) or a naso-gastric (NG) tube.

In alternative embodiments, methods or compositions of the inventionformulate or use various formulations, e.g., frozen extracted stoolbacterial material can be suspended as a flavoured drink or put down anND or an NG tube or inserted as an enema.

In alternative embodiments, extracted bacteria—the ‘wild types’ arefreeze-dried (optionally, after partial, substantial or completepurification and isolation) and formed into powder; they then can beingested, e.g., as enteric-coated capsules, tablets, solutions and thelike.

In alternative embodiments, these ‘products’ of the invention areinitially taken, infused or administered daily, then less and lessfrequently, and in some embodiments, ultimately once every few weeks ormonthly.

In alternative embodiments cultured bacteria can be used in addition toor with the partial, substantial or completely purified or isolatedfecal flora. For example, in one embodiment the first administration orthe initial daily formulations comprise only partial, substantial orcompletely no purified or isolated fecal flora; while in otherembodiments the first administration or the initial daily formulationscomprise both partial, substantial or completely purified or isolatedfecal flora and cultured bacteria, e.g., cultured probiotic bacteria. Inalternative embodiments, the less frequent formulations or dosages(which can be stepwise in small or larger intervals, or periodicintervals, or intervals as determined by the physician or veterinarianaccording to rate of improvement, and the like) comprise only partial,substantial or completely purified or isolated fecal flora; while inother embodiments comprise both partial, substantial or completelypurified or isolated fecal flora and cultured bacteria; or in otherembodiments comprise only cultured bacteria, e.g., cultured probioticbacteria.

In alternative embodiments, to achieve a desired effect or therapeuticoutcome, the cultured bacteria is a Bacteroides and/or Firmicutesspecies and/or Bacillus thuringiensis, which may be directly isolatedfrom a donor, or can come from a pure culture, and the like. Inalternative embodiments, a delivery of an enhanced amount of one or morefecal flora (e.g., bacterial) species is used, e.g., the deliveredproduct is enhanced with (is “spiked” with”) one or more additionalspecies, e.g., a Bacteroides and/or Firmicutes species and/or Bacillusthuringiensis.

In alternative embodiments, for adequate efficacy as to be determined bythe skilled artisan, the formulations are introduced daily, or notdaily—but instead recurrently for prolonged periods of time, e.g., inmuch higher doses. In alternative embodiments, the repeated or multipleinfusion, administration or implantation protocols comprise infusionsdone daily for about the first 10 days, and subsequently a second dailydifferent dosage or formulation for about 10 days, and optionally asubsequent different third daily; then optionally a different fourthdaily, weekly, or monthly dosage or formulation, and then optionallymaintaining different dosages or formulations for a further daily,weekly or monthly delivery or infusion until the histology reversestowards normality or other treatment parameter or goal is achieved;e.g., for the treatment of Irritable Bowel Syndrome, colitis such asUlcerative Colitis or Crohn's Colitis, constipation, autism,degenerative neurological diseases such as Multiple Sclerosis (MS),Parkinson's Disease (PD), Myoclonus Dystonia, Rheumatoid Arthritis,Chronic Fatigue Syndrome, obesity, diabetes, type II diabetes,Idiopathic thrombocytopenic purpura (ITP), autoimmune diseases, chronicurticaria and/or insomnia or chronic insomnia, Grand mal seizures orpetit mal seizures.

In alternative embodiments, the repeated or multiple infusion,administration or implantations are done with: a first formulation dailyfor the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days;a second dosage or formulation daily for a subsequent 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 days or more; then optionally a thirdsubsequent dosage or formulation daily (e.g., for a subsequent 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days or more); thenoptionally a fourth dosage or formulation daily or weekly (e.g., for asubsequent 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days,weeks, months or more); and optionally then maintaining weekly ormonthly infusions until e.g., the histology reverses towards normality,or other appropriate parameter for treatment or recovery; e.g., fortreatment of Irritable Bowel Syndrome, colitis such as UlcerativeColitis or Crohn's Colitis, constipation, autism, degenerativeneurological diseases such as Multiple Sclerosis (MS), Parkinson'sDisease (PD), Myoclonus Dystonia, Rheumatoid Arthritis, Chronic FatigueSyndrome, obesity, diabetes, type II diabetes, Idiopathicthrombocytopenic purpura (ITP), autoimmune diseases, chronic urticariaand/or insomnia or chronic insomnia, Grand mal seizures or petit malseizures. One of skill in the art, e.g., a physician or veterinarian,can assess the individual's improvement and determine the exact,appropriate dosage or frequency of administration in this “repeatadministration” embodiment of the invention.

In alternative embodiments, these exemplary protocols also can be usedfor infusing or ingesting cultured probiotic bacteria that would beswept down the bowel in waves so as to address the issue of the biofilmsspores, dormant forms and intracellular bacteria.

In summary, in alternative embodiments, the invention providescompositions and methods for treating, stabilizing, or ameliorating gutflora infections that are difficult to permanently reverse, or fortreating or ameliorating conditions characterised by or related to gutflora infections that are difficult to permanently reverse or control,by multiple, repeated infusions of fecal flora, as described herein. Inalternative embodiments the fecal microbiota transplant compositions andmethods of the invention are effective in the more difficult conditionslisted above in addition to conditions where a Clostridium, e.g., C.difficile, is the infective agent. In alternative embodiments, repeatedor recurrent infusions are the key to obtaining a cure, a stabilizationor a prolonged remission.

Devices for Delivering Compositions of the Invention

The invention also provides devices for delivering compositions of theinvention, e.g., an exemplary delivery device is illustrated in FIG. 1B.In alternative embodiments, a device of the invention also can compriseor consist of:

(b) (1) a bag or container comprising an exit aperture operablyconnected to the proximal end of a flexible tube or equivalent,

wherein the bag or container is optionally made of a material imperviousto a gas or to oxygen,

and optionally the bag or container is made of a flexible material, or apolyethylene terephthalate polyester film-comprising (or aMYLAR™-comprising) material,

and optionally the bag or container is an (IV-like) intravenous-likebag,

and optionally the bag or container has an attachment that will allowthe bag to be hung on a stand, e.g., to be positioned/hung above anendoscope,

and optionally the bag or container is operably connected via an open orclose valve or equivalent to a negative pressure device that can removeall gas or air from the bag,

and optionally the bag or container is operably connected via an open orclose valve or equivalent to a fluid source or storage container forflushing out the bag through the exit aperture, and optionally the fluidsource or storage container is under positive pressure,

and optionally the flexible tube or equivalent comprises at least oneclip or close 25 valve or one way valve to prevent backwash of materialfrom distal to proximal portions of the tube, or from the tube back tothe bag or container;

(2) an open or close valve or equivalent or an obdurator screwtop at thedistal end of the flexible tube or equivalent,

and optionally a Luer lock tip for attachment to a colonoscope or anendoscopic Luer lock port or equivalent, wherein optionally the Luerlock tip is built into the valve, or is separate from the valve,

and optionally an enema tube tip for attachment to an enema tube ordevice or equivalent, wherein optionally the enema tube tip is builtinto the valve, or is separate from the valve,

and optionally further comprising a safety device or safety clip toclose the distal 5 aperture in case the valve or Luer lock tip, or enematip, is lost (flies off) under pressure; and

(3) a pump, or a hand pump, for moving material in the bag or containerthrough the flexible tube or equivalent and out the distal end or outthe open or close valve or equivalent.

In alternative embodiments, a device of the invention further comprisesa treated or untreated fecal flora, or a partially, substantially orcompletely isolated fecal flora, or a composition of the invention,e.g., a partially, substantially or completely isolated or purifiedfecal flora, or a composition comprising a fecal flora substantially orcompletely purified of non-fecal floral fecal material, and optionallyfurther comprising an excipient, or a fluid, a saline, a buffer, abuffering agent or a media, or a fluid-glucose-cellobiose agar (RGCA)media.

In one embodiment, the invention provides a bag or container comprisinga treated or untreated fecal flora, or a partially, substantially orcompletely isolated fecal flora, or a composition of the invention,e.g., a partially, substantially or completely isolated or purifiedfecal flora, or a composition comprising a fecal flora substantially orcompletely purified of non-fecal floral fecal material, and optionallyfurther comprising an excipient, or a fluid, a saline, a buffer, abuffering agent or a media, or a fluid-glucose-cellobiose agar (RGCA)media, wherein the bag or container is structurally the same as orsimilar to a bag or container of a device of the invention, e.g., a bagor container comprising an exit aperture operably connected to theproximal end of a flexible tube or equivalent, etc., as describedherein.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1: Exemplar Methods of the Invention

One exemplary procedure of the invention involves a 5- to 10-daytreatment with enemas comprising a treated or isolated fecal bacterialflora of the invention initially derived from a healthy donor.Alternatively, patients can recover after just one treatment.

In one embodiment, the best choice for donor is a close relative who hasbeen tested for a wide array of bacterial and parasitic agents. Theenemas are prepared and administered in a hospital environment to ensureall necessary precautions. An exemplary probiotic infusion of theinvention can also be administered through a nasogastric tube,delivering the bacteria directly to the small intestine. These twomethods can be combined to achieve a desired result. Regular checkupsshould be required up to a year following the procedure.

In one embodiment an autologous fecal sample is provided by a patientbefore a medical treatment, and it is stored in a refrigerator,lyophilized or freeze-dried or equivalent. Should the patientsubsequently develop an infection, e.g., a C. difficile infection, thesample is prepared (extracted) with saline and filtered. The filtratecan be freeze-dried and the resulting solid enclosed in a capsule, e.g.,an enteric coated capsule. Administration of the capsules can restorethe patient's own colonic flora and combat the infection, e.g., the C.difficile. In one embodiment, samples are delivered into the duodenumvia a nasal probe.

In one embodiment, a method of the invention comprises the collectionfrom healthy donors of fresh, human flora (stool), bringing it to acentralized institution, processing it in such a fashion that it will begiven prolonged life, checking for pathogens, maintaining temperaturecontrol to reduce metabolic activity of the bacteria and controlling foroxygen-shock, developing a storage facility of the homogenized,standardized flora, and shipping the flora out to distant hospitals totreat patients with e.g. acute pseudo membranous colitis, severe C.difficile infection, septicaemia or other comparable conditions.

In one embodiment, the product of the invention is a modified stoolcomposition. The stool needs to be collected and promptly placed into ananaerobic container which extracts air, possibly with substances thatadsorb and absorb oxygen or can be evacuated and filled with nitrogen orother gas which is either inert or will not damage anaerobic flora. Ithas to be held in an aesthetically acceptable container which will notleak the stool nor the gas which is producing the anaerobic situation.Once delivered to the central ‘bank’ the stool can be stored in a coldroom to slow down metabolism but not be frozen to prevent the waterexpansion-destruction of the bacterial cells contained in stool.

In one embodiment, either antioxidants and/or substances such asglycerol are added to help stabilize the bacteria in the cold andprevent them from becoming destroyed during storage and duringtransport.

In one embodiment the product is stored/contained as (in) a volume ofbetween about 10 cc and 3 liters of stool. In one embodiment it isstored in a (as a) 300 cc container (or amount) and held in appropriateoxygen-resistant material, e.g., a plastic, an oxygen-resistant or gasimpervious polyethylene terephthalate polyester film (e.g., inmetallized form), or a metallized MYLAR™, or an aluminized MYLAR™, whichcan be attached to a pump through a giving set that will be attached tothe colonoscope and administered through a colonoscope into a distalsmall bowel or into the upper colon/terminal ileum, to overcomeClostridium difficile infection.

Central Flora Supply Institution or “Bank”

in one embodiment, an institution functions to supply the human flora inthe following manner:

1. Stool will be collected in special containers and held coolanaerobically until it arrives at the central flora processing unit.

2. In a processing unit special additives will be added includingglycerol, possibly antioxidants and other special preservatives and keptcool, homogenized and dispensed into appropriate intravenous-like bagsbut with somewhat thicker product such as a gas impervious polyethyleneterephthalate polyester film, or an aluminized MYLAR™. This will preventoxygen being transferred, nitrogen escaping and the smell being detectedby administering staff. The bags will then be kept stored at atemperature that does not allow bacteria to freeze and be ready fortransport in coolers to hospitals that will be carrying out the faecaltransplantation.

3. The bag will be supplied with an attached giving set, so that it doesnot have to be handled by hospital staff. There will be attached to it a‘blood type’ pump, with one way valve. On the (IV-like) intravenous-likebag there will be attachments that will be able to allow the bag to behung on an IV fluids stand and be positioned/hung above the endoscope.The endoscopist will then take off the obdurator screwtop and attach tothe Luer lock tip onto the endoscopic Luer lock port to be infusedthrough the biopsy forceps channel at the tip of the colonoscope orendoscope. A safety device would be attached in case the tip flies offunder pressure. The air will then be bled from the tube as the productis allowed to run down the ‘giving set’ with pressure mechanisms alongthe giving set, with air bled, and then stool only will be administeredusing the administering pump into the patient's colon and flushed, forexample with some saline.

4. The endoscopist would then withdraw the colonoscope, turn the patient‘head down/legs up’ to allow air and liquid to be absorbed and preventthe patient from undergoing defecation too early. This will allow thebacteria to re-gain temperature, start attaching themselves to the bowelwall as described e.g., by Grehan et al: J of Clinical Gastroenterology,September 2010.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-43. (canceled)
 44. A pharmaceutical composition comprising a mixtureof (a) a microbiota of a stool sample of a human donor; and (b) acultured bacterial strain.
 45. The pharmaceutical composition of claim44, wherein the cultured bacterial strain comprise a member of thephylum Firmicutes.
 46. The pharmaceutical composition of claim 44,wherein the cultured bacterial strain comprises a member of the genusBacteroides.
 47. The pharmaceutical composition of claim 44, wherein themicrobiota is derived from a stool sample of a healthy human donor. 48.The pharmaceutical composition of claim 47, wherein the microbiota ismixed with fiber from the stool sample.
 49. The pharmaceuticalcomposition of claim 47, wherein the microbiota is a substantiallycomplete microbiota of the stool sample.
 50. The pharmaceuticalcomposition of claim 44, wherein the cultured bacterial strain isderived from a healthy human donor.
 51. The pharmaceutical compositionof claim 44, wherein the composition comprises multiple culturedbacterial strains.
 52. The pharmaceutical composition of claim 44,wherein the cultured bacterial strain comprises anaerobic bacteria. 53.The pharmaceutical composition of claim 44, for use in treating autismin a subject in need thereof.
 54. The pharmaceutical composition ofclaim 44, wherein the composition further comprises an antioxidant. 55.The pharmaceutical composition of claim 44, wherein at least one of themicrobiota and the cultured bacterial strain is lyophilized.
 56. Thepharmaceutical composition of claim 44, wherein the microbiota and thecultured bacterial strain are from different sources.
 57. A methodcomprising mixing a cultured bacterial strain with a microbiota of astool sample of a human donor.
 58. The method of claim 57, wherein theculturing of the cultured bacterial strain is under anaerobicconditions.
 59. The method of claim 57, wherein the cultured bacterialstrain is lyophilized.
 60. The method of claim 57, wherein the humandonor is a healthy human donor.
 61. The method of claim 57, furthercomprising mixing a stabilizing agent with the supplemental isolatedbacterial strain and the microbiota of a stool sample of a human donor.62. The method of claim 57 further comprising providing the mixture to asubject in need thereof having autism.
 63. The method of claim 57,wherein the microbiota and the cultured bacterial strain are fromdifferent sources.